The phenomenon of programmed cell death, or "apoptosis," is known to be involved in and important to the normal course of a wide variety of developmental processes, including immune and nervous system maturation. Apoptosis also plays a role in adult tissues having high cell turnover rates (Ellis, R. E., et al., Annu. Rev. Cell. Biol. 7: 663-698 (1991); Oppenheim, R. W., Annu. Rev. Neurosci. 14: 453-501 (1991); Cohen, J. J., et al. Annu. Rev. Immunol. 10: 267-293 (1992); Raff, M. C., Nature 356: 397-400 (1992)). A number of different physiological signals normally activate programmed cell death in these contexts, but non-physiological insults, such as irradiation and exposure to drugs which damage DNA, also can trigger apoptosis (Eastman, A., Cancer Cells 2: 275-280 (1990); Dive, C., et al., Br. J. Cancer 64: 192-196 (1991); Lennon, S. V., et al., Cell Prolif. 24: 203-214 (1991)).
In addition to its role in development, apoptosis has been implicated as an important cellular safeguard against tumorigenesis (Williams, G. T., Cell 65: 1097-1098 (1991); Lane, D. P., Nature 362: 786-787 (1993)). Under certain conditions, cells die by apoptosis in response to high-level or deregulated expression of oncogenes (Askew, D., et al., Oncogene 6: 1915-1922 (1991); Evan, G. I., et al., Cell 69: 119-128 (1992); Rao, L., et al., Proc. Natl. Acad. Sci. USA 89: 7742-7746 (1992); Smeyne, R. J., et al., Nature 363: 166-169 (1993); Tanaka, S., et al., Cell 77: 829-839 (1994); Wu, X., et al., Proc. Natl. Acad. Sci. USA 91: 3602-3606 (1994)). Suppression of the apoptotic program, by a variety of genetic lesions, may contribute to the development and progression of malignancies. This is well illustrated by the frequent mutation of the p53 tumor suppressor gene in human tumors (Levine, A. J., et al., Nature 351: 453-456 (1991)). Wild-type p53 is required for efficient induction of apoptosis following DNA damage (Clarke, A. R., et al., Nature 362: 849-852 (1993); Lowe, S. W., et al., Cell 74: 957-967 (1993); Lowe, S. W., et al., Nature 362: 847-849 (1993)) and cell death induced by constitutive expression of certain oncogenes (Debbas, M., et al., Genes & Dev. 7: 546-554 (1993); Hermeking, H., et al., Science 265: 2091-2093 (1994); Tanaka, S., et al., Cell 77: 829-839 (1994); Wu, X., et al., Natl. Acad. Sci. USA 91: 3602-3606 (1994)). The cytotoxicity of many commonly used chemotherapeutic agents is mediated by wild-type p53 (Lowe, S. W., et al., Cell 74: 957-967 (1993); Fisher, D. E., Cell 78: 539-542 (1994)). Thus, loss of p53 function may contribute to the clinically significant problem of drug resistant tumor cells emerging following chemotherapy regimens.
The expression product of the bcl-2 oncogene functions as a potent suppressor of apoptotic cell death (McDonnell, T. J., et al., Cell 57: 79-88 (1989); Hockenbery, D., et al., Nature 348: 334-336 (1990)). Constitutive Bcl-2 expression can suppress apoptosis triggered by diverse stimuli, including growth factor withdrawal oncogene expression, DNA damage, and oxidative stress (Vaux, D. L., et al., Nature 335: 440-442 (1988); Sentman, C. L., et al., Cell 67: 879-888 (1991); Strasser, A., et al., Cell 67: 889-899 (1991); Fanidi, A., et al., Nature 359: 554-556 (1992); Hockenbery, D. M., et al., Cell 75: 241-251 (1993)). There is also conservation of Bcl-2 function across species. For example, the ced-9 gene of the nematode C. elegans appears to be a structural and functional homolog of bcl-2 (Hengartner, M. O., et al., Cell 76: 665-676 (1994)) and bcl-2 can complement ced-9 mutations in transgenic animals (Vaux, D. L., et al., Science 258: 1955-1957 (1991)). These observations suggest that Bcl-2 is intimately connected with an evolutionarily conserved cell death program.
It is known that bcl-2 is a member of a family of related genes, at least some of which also modulate apoptosis. Of these, bcl-x bears the highest degree of homology to bcl-2, and is differentially spliced to produce a long form, termed bcl-x.sub.L, and a shorter form, bcl-x.sub.S, harboring an internal deletion (Boise, L. H., et al., Cell 74: 597-608 (1993)). Bcl-x.sub.L functions to suppress apoptosis, whereas the deleted form, Bcl-x.sub.S, inhibits the protection against cell death provided by Bcl-2 expression. A second Bcl-2 homolog, Bax, forms heterodimers with Bcl-2 (Oltvai, Z. N., et al., Cell 74: 609-619 (1993)) and has been shown to counteract Bcl-2 and accelerate apoptosis. Mutational analysis of Bcl-2 has suggested that the interaction with Bax is required for Bcl-2 to function as an inhibitor of cell death (Yin, X.-M., et al., Nature 369: 321-323 (1994)).
The isolation and characterization of a bcl-2 related gene, termed bak, is described in U.S. application Ser. No. 08/321,071, filed 11 Oct. 1994, U.S. Pat. No. 5,672,686 which is a continuation-in-part of U.S. application Ser. No. 08/287,427, filed 9 Aug. 1994, abandoned (bak is referred to therein as bcl-y), the disclosures of which are incorporated herein by reference. Ectopic Bak expression accelerates the death of an IL-3 dependent cell line upon cytokine withdrawal, and opposes the protection against apoptosis afforded by Bcl-2. In addition, enforced expression of Bak is sufficient to induce apoptosis of serum deprived fibroblasts, raising the possibility that Bak directly activates, or is itself a component of, the cell death machinery.
The known cellular Bcl-2 related genes, where analyzed, have distinct patterns of expression and thus may function in different tissues. While Bcl-2 expression appears to be required for maintenance of the mature immune system, it is desirable to identify other genes which may govern apoptotic cell death in other lineages. In addition, the identification of particular regions or domains of the proteins encoded by such genes may provide a basis for understanding their structural and functional characteristics and allow the development of valuable diagnostics and therapeutics. For example, the identification of agents capable of restoring or inducing apoptosis in tumor cells (in which loss of p53 tumor suppressor gene function may be implicated in tumorigenesis and in clinically significant drug resistance) would be of significant therapeutic value, particularly where such restoration or induction was independent of p53 function. Similarly, the development of agents capable of counteracting the anti-apoptotic function of oncogenes such as bcl-2, the activation of which is implicated in tumorigenesis (e.g., lymphoma) and in chemotherapeutic drug resistance, would be of great potential value.